Poly(L-lysine isolphthalamide) (PLP) polymers with hydrophobic pendant chains

Abstract

The present disclosure relates to the provision of novel biodegradable amphiphilic peptides and peptide analogues based derivatives comprising hydrophobic chains and their use in the permeabilization of mammalian cells and delivery of agents, for example therapeutic agents, imaging agents and cell preservation agents.

The invention claimed is: 
     
       1. A poly(lysine isophthalamide) derivative comprising general formula (I): 
       
         
           
           
               
               
           
         
         wherein R comprises 
         NR 1 R 2  or OH wherein at least one of R are NR 1 R 2 ; 
         R 1  and R 2  each independently comprises: 
         H; 
         C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, C 6-10  aryl or C 5-10  heteroaryl; wherein 
         Alkyl, alkenyl and alkynyl groups R 1  and R 2  are optionally substituted with one or more substituents selected from halo, cyano, nitro, diazonium, —OP(O)OR 3 OR 4 , —PR 3 R 4 , NR 3 R 4 , ═NR 3 , ═O, C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 3-7  cycloalkyl, C 3-10  heterocyclyl, C 6-14  aryl or C 4-14  heteroaryl;
 wherein cycloalkyl, heterocyclyl, aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 , aryl substituted with R 3  and heteroaryl substituted with R 3  and, where chemically appropriate, ═O; and 
 wherein each of R 3  and R 4  is independently H or C 1-10  alkyl, C 2-10  alkenyl, C 2-10  alkynyl, C 6-10  aryl; 
 
         aryl and heteroaryl groups R 1  and R 2  are optionally substituted with one or more substituents selected from C 1-16  alkyl, halo, cyano, nitro, diazonium, —OP(O)OR 3 OR 4 , —PR 3 R 4 , NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl, C 4-14  heteroaryl or S—CH 2 C(O)NR 5 R 6 ;
 wherein alkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; 
 wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; 
 
         wherein R 3  and R 4  are as defined above and R 5  and R 6  are each independently H, C 1-6  alkyl optionally substituted with OR 3  or halo or C 6-14  aryl optionally substituted with C 1-6  alkyl, OH, O(C 1-6  alkyl) or O—C 6-14  aryl; or 
         R 1  and R 2  together with the nitrogen atom to which they are attached form a 5-12-membered heterocyclic ring optionally containing one or more additional heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected from C 1-16  alkyl, C 1-16  haloalkyl, halo, cyano, nitro, diazonium, ═O, —OP(O)OR 3 R 4 , —PR 3 R 4 , NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl or C 4-14  heteroaryl;
 wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 , 
 wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; 
 
         wherein R 3  and R 4  are as defined above; and 
         n≥4. 
       
     
     
       2. A poly(lysine isophthalamide) derivative according to  claim 1  wherein
 R 1  and R 2  each independently comprises: 
 H; 
 C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl group optionally substituted with one or more substituents selected from halo, cyano, nitro, diazonium, —OP(O)OR 3 OR 4 , —PR 3 R 4 , NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl or C 4-14  heteroaryl,
 wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; and wherein each of R 3  and R 4  is independently H or C 1-10  alkyl, C 2-10  alkenyl, C 2-10  alkynyl, C 6-10  aryl; 
 
 C 6-10  aryl optionally substituted with one or more substituents selected from C 1-16  alkyl, C 1-16  haloalkyl, halo, cyano, nitro, diazonium, —OP(O)OR 3 OR 4 , —PR 3 R 4 , NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl or C 4-14  heteroaryl,
 wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; 
 wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ;
 wherein R 3  and R 4  are as defined above; or 
 
 
 R 1  and R 2  together with the nitrogen atom to which they are attached to form a 5-12-membered heterocyclic ring optionally containing one or more additional heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected from C 1-16  alkyl, C 1-16  haloalkyl, halo, cyano, nitro, diazonium, —OP(O)OR 3 OR 4 , —PR 3 R 4 , NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl or C 4-14  heteroaryl;
 wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 , 
 wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , azide, OP(O)OR 3 OR 4 , —PR 3 R 4 ; 
 wherein R 3  and R 4  are as defined above; and n≥4. 
 
 
     
     
       3. A poly(lysine isophthalamide) derivative according to  claim 1  wherein R 1  is as defined in  claim 1  and R 2  is C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, C 6-10  aryl or C 5-10  heteroaryl, any of which is optionally substituted as defined in  claim 1 . 
     
     
       4. A poly(lysine isophthalamide) derivative according to  claim 3 , wherein R 1  is H, C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, any of which may optionally be substituted as defined in  claim 1  and R 2  is C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, any of which may optionally be substituted as defined in  claim 1 . 
     
     
       5. A poly(lysine isophthalamide) derivative according to  claim 4  wherein
 R 1  is H or C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, any of which is unsubstituted or is substituted with F, Cl, OH, SH, methoxy or ethoxy; and 
 R 2  is C 6-30  alkyl, C 6-30  alkenyl or C 6-30  alkynyl, any of which is unsubstituted or is substituted with F, Cl, OH, SH, methoxy or ethoxy. 
 
     
     
       6. A poly(lysine isophthalamide) derivative according to  claim 5  wherein R 1  is H or unsubstituted C 6-30  alkyl, unsubstituted C 6-30  alkenyl or unsubstituted C 6-30  alkynyl; and R 2  is unsubstituted C 6-30  alkyl, unsubstituted C 6-30  alkenyl or unsubstituted C 6-30  alkynyl. 
     
     
       7. A poly(lysine isophthalamide) derivative according to  claim 5 , wherein R 1  is H and R 2  is unsubstituted C 7-18  alkyl. 
     
     
       8. A poly(lysine isophthalamide) derivative according to  claim 7 , wherein each of R 1  and R 2  is independently unsubstituted C 7-18  alkyl. 
     
     
       9. The poly(lysine isophthalamide) derivative according to  claim 7  wherein R 2  is n-heptyl, n-decyl, n-tetradecyl or n-octadecyl. 
     
     
       10. The poly(lysine isophthalamide) derivative according to  claim 9  wherein said R 2  is n-decyl. 
     
     
       11. The poly(lysine isophthalamide) derivative according to  claim 1  wherein between 0.1-99% of the moieties R are NR 1 R 2 . 
     
     
       12. The poly(lysine isophthalamide) derivative according to  claim 11  wherein 3-18% of the moieties R are NR 1 R 2 . 
     
     
       13. The poly(lysine isophthalamide) derivative according to  claim 2  wherein each of R 1  and R 2  is C 7-18  alkyl, C 7-18  alkenyl or C 7-18  alkynyl, any of which may optionally be substituted as defined in  claim 2 . 
     
     
       14. The poly(lysine isophthalamide) derivative according to  claim 13  wherein each of R 1  and R 2  is C 7 -alkyl, C 8 -alkyl, C 10  alkyl, C 14  alkyl or C 18  alkyl, any of which may be optionally be substituted according to  claim 2 . 
     
     
       15. The poly(lysine isophthalamide) derivative according to  claim 1  wherein each of R 1  and R 2  is optionally substituted with one or more substituents selected from halo, cyano, nitro, azo, diazonium, phosphate, phosphate ester, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , C(O)SR 3 , C(O)NR 3 R 4 , azide, C 6-14  aryl or C 4-14  heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-10  alkyl, C 1-10  haloalkyl, C 2-10  alkenyl, C 2-10  alkynyl, halo, cyano or nitro, NR 3 R 4 , C(O)OR 3 , OR 3 , SR 3 , RN 3 , phosphate, phosphate ester and wherein each of R 3  and R 4  is independently H or C 1-10  alkyl, C 2-10  alkenyl, C 2-10 alkynyl. 
     
     
       16. The poly(lysine isophthalamide) derivative according to  claim 2  wherein each of R 1  and R 2  is optionally substituted with one or more substituents selected from halo, cyano, nitro, NR 3 R 4 , C(O)OR 3 OR 3 SR 3 , C 6-10  aryl or heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C 1-4  alkyl, C 1-4  haloalkyl, halo, cyano or nitro; and wherein each of R 3  and R 4  is independently H or C 1-6  alkyl. 
     
     
       17. The poly(lysine isophthalamide) derivative according to  claim 1  wherein said peptide is associated, either directly or indirectly with an agent for intracellular delivery to a cell. 
     
     
       18. The poly(lysine isophthalamide) derivative according to  claim 17  wherein said agent is covalently or non-covalently associated with said poly(lysine isophthalamide) derivative. 
     
     
       19. The poly(lysine isophthalamide) derivative according to  claim 18  wherein said agent is a therapeutic agent. 
     
     
       20. The poly(lysine isophthalamide) derivative according to  claim 18  wherein said agent is an imaging agent. 
     
     
       21. The poly(lysine isophthalamide) derivative according to  claim 18  wherein said agent is a cell preservation agent. 
     
     
       22. A process for the preparation of poly(lysine isophthalamide) derivative according to  claim 1  comprising the steps
 i) polymerization of aqueous lysine methyl ester.2HCl with an equivalent amount of isophthaloyl chloride in acetone and subsequent hydrolysis in DMSO with ethanolic sodium hydroxide, and 
 ii) conjugation of R, wherein R comprises NR 1 R 2  and is defined as above onto the polymer backbone via dicyclohexylcarboiimide/dimethylaminopyridine (DCC/DMAP) coupling; or
 conjugation of R is via 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) coupling. 
 
 
     
     
       23. A composition comprising a poly(lysine isophthalamide) derivative according to  claim 1 . 
     
     
       24. The composition according to  claim 23  wherein the agent is a therapeutic agent and the composition is a pharmaceutical composition including a pharmaceutically acceptable carrier. 
     
     
       25. The composition according to  claim 23  wherein said composition comprises mammalian cells or a collection of mammalian cells. 
     
     
       26. A composition according to  claim 23  for use in the delivery of at least one agent to a mammalian cell, cellular aggregate, tissue or organ. 
     
     
       27. An in vitro or ex vivo method to deliver an agent to a cell comprising:
 i) contacting cells or a cellular aggregate, tissue or organ comprising cells with an effective amount of a composition according to  claim 23 ; and 
 ii) incubating said cell, cellular aggregate, tissue or organ to allow permeabilization of said mammalian cells or cellular aggregate, tissue or organ comprising cells thereby delivering said agent. 
 
     
     
       28. The method according to  claim 27  wherein said cell is a mammalian cell. 
     
     
       29. An in vitro or ex vivo method for the preservation of a mammalian cell, cellular aggregate, tissue or organ comprising the steps:
 i) providing a preparation comprising a mammalian cell preparation, mammalian cellular aggregate, tissue or organ and a composition according to  claim 23 ; 
 ii) incubating said preparation to permeabilize the mammalian cell membranes of said mammalian cell, cellular aggregate, tissue or organ; and 
 iii) contacting said permeabilized cell, cellular aggregate, tissue or organ with one or more preservation agents. 
 
     
     
       30. The method according to  claim 29  wherein said preservation agent is a sugar.